Xylofuranosly-containing nucleoside phosphoramidites and polynucleotides

ABSTRACT

Novel xylo nucleoside or xylo nucleotide analogs, polynucleotides comprising xylo nucleotide substitution, processes for their synthesis and incorporation into polynucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of Jasenka Matulic-Adamic, etal., U.S. Provisional Application 60/056,808, entitled“Xylofiranosyl-Containing Nucleoside Phosphoramidites andPolynucleotides”, filed Aug. 22, 1997. This application is herebyincorporated herein by reference in its entirety, including any drawingsand figures.

BACKGROUND OF THE INVENTION

[0002] This invention relates to novel nucleoside or nucleotide analogs,and processes for their synthesis and incorporation intopolynucleotides.

[0003] The following is a brief description of nucleoside analogs. Thissummary is not meant to be complete but is provided only for anunderstanding of the invention that follows. This summary is not anadmission that all of the work described below is prior art to theclaimed invention.

[0004] Nucleoside modifications of bases and sugars, have beendiscovered in a variety of naturally occurring RNA (e.g., tRNA, mRNA,rRNA; reviewed by Hall, 1971 The Modified Nucleosides in Nucleic Acids,Columbia University Press, New York; Limbach et al., 1994 Nucleic AcidsRes. 22, 2183). In an attempt to understand the biological significance,structural and thermodynamic properties, and nuclease resistance ofthese nucleoside modifications in nucleic acids, several investigatorshave chemically synthesized nucleosides, nucleotides andphosphoramidites with various base and sugar modifications andincorporated them into oligonucieotides.

[0005] Uhlmann and Peyman, 1990, Chem. Reviews 90, 543, review the useof certain nucleoside modifications to stabilize antisenseoligonucleotides.

[0006] Usman et al., International PCT Publication Nos. WO/93/15187; andWO 95/13378; describe the use of sugar, base and backbone modificationsto enhance the nuclease stability of enzymatic nucleic acid molecules.

[0007] Eckstein et al., International PCT Publication No. WO 92/07065describe the use of sugar, base and backbone modifications to enhancethe nuclease stability of enzymatic nucleic acid molecules.

[0008] Grasby et al., 1994, Proc. Indian Acad. Sci., 106, 1003, reviewthe “applications of synthetic oligoribonucleotide analogues in studiesof RNA structure and function”.

[0009] Eaton and Pieken, 1995, Annu. Rev. Biochem., 64, 837, reviewsugar, base and backbone modifications that enhance the nucleasestability of RNA molecules.

[0010] Rosemeyer et al., 1991, Helvetica Chem. Acta, 74, 748, describethe synthesis of 1-(2′-deoxy-β-D-xylofuranosyl) thymine-containingoligodeoxynucleotides.

[0011] Seela et al., 1994, Helvetica Chem. Acta, 77, 883, describe thesynthesis of 1-(2′-deoxy-β-D-xylofuranosyl) cytosine-containingoligodeoxynucleotides.

[0012] Seela et al., 1996, Helvetica Chem. Acta, 79, 1451, describe thesynthesis xylose-DNA containing the four natural bases.

[0013] The references cited above are distinct from the presentlyclaimed invention since they do not disclose and/or contemplate thesynthesis of xylofuranosyl nucleoside phosphoamidites andpolynucleotides comprising such nucleotide modifications of the instantinvention.

SUMMARY OF THE INVENTION

[0014] This invention relates to a compound having the Formula I:

[0015] wherein, R₁ is OH, O—R₃, where R₃ is independently a moietyselected from a group consisting of alkyl, alkenyl, alkynyl, aryl,alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester; C—R₃,where R₃ is independently a moiety selected from a group consisting ofalkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclicaryl, amide and ester; halo, NHR₄ (R₄=alkyl (C1-22), acyl (C1-22),substituted or unsubstituted aryl), or OCH₂SCH₃ (methylthiomethyl),ONHR₅ where R₅ is independently H, aminoacyl group, peptidyl group,biotinyl group, cholesteryl group, lipoic acid residue, retinoic acidresidue, folic acid residue, ascorbic acid residue, nicotinic acidresidue, 6-aminopenicillanic acid residue, 7-aminocephalosporanic acidresidue, alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide or ester, ON═R₆, where R₆ is independentlypyridoxal residue, pyridoxal-5-phosphate residue, 13-cis-retinalresidue, 9-cis-retinal residue, alkyl, alkenyl, alkynyl, alkylaryl,carbocyclic alkylaryl, or heterocyclic alkylaryl; B is independently anucleotide base or its analog or hydrogen; X is independently aphosphorus-containing group; and R₂ is independently blocking group or aphosphorus-containing group.

[0016] Specifically, an “alkyl” group refers to a saturated aliphatichydrocarbon, including straight-chain, branched-chain, and cyclic alkylgroups. Preferably, the alkyl group has 1 to 12 carbons. More preferablyit is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4carbons. The alkyl group may be substituted or unsubstituted. Whensubstituted the substituted group(s) is preferably, hydroxy, cyano,alkoxy, NO₂ or N(CH₃)₂, amino, or SH.

[0017] The term “alkenyl” group refers to unsaturated hydrocarbon groupscontaining at least one carbon-carbon double bond, includingstraight-chain, branched-chain, and cyclic groups. Preferably, thealkenyl group has 1 to 12 carbons. More preferably it is a lower alkenylof from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenylgroup may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, NO₂,halogen, N(CH₃)₂, amino, or SH.

[0018] The term “alkynyl” refers to an unsaturated hydrocarbon groupcontaining at least one carbon-carbon triple bond, includingstraight-chain, branched-chain, and cyclic groups. Preferably, thealkynyl group has 1 to 12 carbons. More preferably it is a lower alkynylof from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynylgroup may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino or SH.

[0019] An “aryl” group refers to an aromatic group which has at leastone ring having a conjugated π electron system and includes carbocyclicaryl, heterocyclic aryl and biaryl groups, all of which may beoptionally substituted. The preferred substituent(s) on aryl groups arehalogen, trihalomethyl, hydroxyl, SH, cyano, alkoxy, alkyl, alkenyl,alkynyl, and amino groups.

[0020] An “alkylaryl” group refers to an alkyl group (as describedabove) covalently joined to an aryl group (as described above).

[0021] “Carbocyclic aryl” groups are groups wherein the ring atoms onthe aromatic ring are all carbon atoms. The carbon atoms are optionallysubstituted.

[0022] “Heterocyclic aryl” groups are groups having from 1 to 3heteroatoms as ring atoms in the aromatic ring and the remainder of thering atoms are carbon atoms. Suitable heteroatoms include oxygen,sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl,pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionallysubstituted.

[0023] An “amide” refers to an —C(O)—NH—R, where R is either alkyl,aryl, alkylaryl or hydrogen.

[0024] An “ester” refers to an —C(O)—OR′, where R is either alkyl, aryl,or alkylaryl.

[0025] A “blocking group” is a group which is able to be removed afterpolynucleotide synthesis and/or which is compatible with solid phasepolynucleotide synthesis.

[0026] A “phosphorus containing group” can include phosphorus in formssuch as dithioates, phosphoramidites and/or as part of anoligonucleotide.

[0027] In a preferred embodiment, the invention features a process forsynthesis of the compounds of formula I.

[0028] In a preferred embodiment the invention features a process forthe synthesis of a xylofuranosyl nucleoside phosphoramidite comprisingthe steps of: a) oxidation of a 2′ and 5′-protected ribonucleoside witha an oxidant such as chromium oxide/pyridine/aceticanhydride,dimethylsulfoxide/aceticanhydride, or Dess-Martin reagent (periodinane)followed by reduction with a reducing agent such as, triacetoxy sodiumborohydride, sodium borohydride, or lithium borohydride, underconditions suitable for the formation of 2′ and 5′-protectedxylofuranosyl nucleoside; b) phosphitylation under conditions suitablefor the formation of xylofuranosyl nucleoside phosphoramidite.

[0029] In yet another preferred embodiment, the invention features theincorporation of the compounds of Formula I into polynucleotides. Thesecompounds can be incorporated into polynucleotides enzymatically. Forexample by using bacteriophage T7 RNA polymerase, these novel nucleotideanalogs can be incorporated into RNA at one or more positions (Milliganet al., 1989, Methods Enzymol, 180, 51). Alternatively, novel nucleosideanalogs can be incorporated into polynucleotides using solid phasesynthesis (Brown and Brown, 1991, in Oligonucleotides and Analogues: APractical Approach, p. 1, ed. F. Eckstein, Oxford University Press, NewYork; Wincott et al., 1995, Nucleic Acids Res., 23, 2677; Beaucage &Caruthers, 1996, in Bioorganic Chemistry: Nucleic Acids, p 36, ed. S. M.Hecht, Oxford University Press, New York).

[0030] The compounds of Formula I can be used for chemical synthesis ofnucleotide-tri-phosphates and/or phosphoramidites as building blocks forselective incorporation into oligonucleotides. These oligonucleotidescan be used as an antisense molecule, 2-5A antisense chimera, triplexforming oligonucleotides (TFO) or as an enzymatic nucleic acid molecule.The oligonucleotides can also be used as probes or primers for synthesisand/or sequencing of RNA or DNA.

[0031] The compounds of the instant invention can be readily convertedinto nucleotide diphosphate and nucleotide triphosphates using standardprotocols (for a review see Hutchinson, 1991, in Chemistry ofNucleosides and Nucleotides, v.2, pp 81-160, Ed. L. B. Townsend, PlenumPress, New York, USA; incorporated by reference herein).

[0032] The compounds of Formula I can also be independently or incombination used as an antiviral, anticancer or an antitumor agent.These compounds can also be independently or in combination used withother antiviral, anticancer or an antitumor agents.

[0033] By “antisense” it is meant a non-enzymatic nucleic acid moleculethat binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA(protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactionsand alters the activity of the target RNA (for a review see Stein andCheng, 1993 Science 261, 1004).

[0034] By “2-5A antisense chimera” it is meant, an antisenseoligonucleotide containing a 5′ phosphorylated 2′-5′-linked adenylateresidues. These chimeras bind to target RNA in a sequence-specificmanner and activate a cellular 2-5A-dependent ribonuclease which, inturn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad.Sci. USA 90, 1300).

[0035] By “triplex forming oligonucleotides (TFO)” it is meant anoligonucleotide that can bind to a double-stranded DNA in asequence-specific manner to form a triple-strand helix. Formation ofsuch triple helix structure has been shown to inhibit transcription ofthe targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci.USA 89, 504).

[0036] By “enzymatic nucleic acid” it is meant a nucleic acid moleculecapable of catalyzing reactions including, but not limited to,site-specific cleavage and/or ligation of other nucleic acid molecules,cleavage of peptide and amide bonds, and trans-splicing. Such a moleculewith endonuclease activity may have complementarity in a substratebinding region to a specified gene target, and also has an enzymaticactivity that specifically cleaves RNA or DNA in that target. That is,the nucleic acid molecule with endonuclease activity is able tointramolecularly or intermolecularly cleave RNA or DNA and therebyinactivate a target RNA or DNA molecule. This complementarity functionsto allow sufficient hybridization of the enzymatic RNA molecule to thetarget RNA or DNA to allow the cleavage to occur. 100% complementarityis preferred, but complementarity as low as 50-75% may also be useful inthis invention. The nucleic acids may be modified at the base, sugar,and/or phosphate groups. The term enzymatic nucleic acid is usedinterchangeably with phrases such as ribozymes, catalytic RNA, enzymaticRNA, catalytic DNA, catalytic oligonucleotides, nucleozyme, DNAzyme, RNAenzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme orDNA enzyme. All of these terminologies describe nucleic acid moleculeswith enzymatic activity. The specific enzymatic nucleic acid moleculesdescribed in the instant application are not limiting in the inventionand those skilled in the art will recognize that all that is importantin an enzymatic nucleic acid molecule of this invention is that it has aspecific substrate binding site which is complementary to one or more ofthe target nucleic acid regions, and that it have nucleotide sequenceswithin or surrounding that substrate binding site which impart a nucleicacid cleaving activity to the molecule. The sites in such nucleic acidthat can be modified with nucleotides described herein are known in theart, or standard methods can be used to determine useful such sites inother such nucleic acids. See, e.g., Usman, supra.

[0037] By “enzymatic portion” or “catalytic domain” is meant thatportion/region of the ribozyme essential for cleavage of a nucleic acidsubstrate (for example see FIG. 7).

[0038] By “substrate binding arm” or “substrate binding domain” is meantthat portion/region of a ribozyme which is complementary to (i.e., ableto base-pair with) a portion of its substrate. Generally, suchcomplementarity is 100%, but can be less if desired. For example, as fewas 10 bases out of 14 may be base-paired. Such arms are shown generallyin FIG. 1 and 3. That is, these arms contain sequences within a ribozymewhich are intended to bring ribozyme and target together throughcomplementary base-pairing interactions. The ribozyme of the inventionmay have binding arms that are contiguous or non-contiguous and may bevarying lengths. The length of the binding arm(s) are preferably greaterthan or equal to four nucleotides; specifically 12-100 nucleotides; morespecifically 14-24 nucleotides long. If a ribozyme with two binding armsare chosen, then the length of the binding arms are symmetrical (i.e.,each of the binding arms is of the same length; e.g., six and sixnucleotides or seven and seven nucleotides long) or asymmetrical (i.e.,the binding arms are of different length; e.g., six and threenucleotides or three and six nucleotides long).

[0039] In a preferred embodiment, a polynucleotide of the inventionwould bear one or more 2′-hydroxylamino functionalities attacheddirectly to the monomeric unit or through the use of an appropriatespacer. Since oligonucleotides have neither aldehyde nor hydroxylaminogroups, the formation of an oxime would occur selectively using an oligoas a polymeric template. This approach would facilitate the attachmentof practically any molecule of interest (peptides, polyamines,coenzymes, oligosaccharides, lipids, etc.) directly to theoligonucleotide using either aldehyde or carboxylic function in themolecule of interest.

[0040] Advantages of oxime bond formation:

[0041] The oximation reaction proceeds in water

[0042] Quantitative yields

[0043] Hydrolytic stability in a wide pH range (5-8)

[0044] The amphoteric nature of oximes allows them to act either as weakacids or weak bases.

[0045] Oximes exhibit a great tendency to complex with metal ions

[0046] In yet another preferred embodiment, the aminooxy “tether” inoligonucleotides, such as a ribozyme, is reacted with differentcompounds bearing carboxylic groups (e.g. aminoacids, peptides, “cap”structures, etc.) resulting in the formation of oxyamides as shownbelow.

[0047] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof,,and from the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] The drawings will first briefly be described.

[0049] Drawings

[0050]FIG. 1 shows the secondary structure model for seven differentclasses of enzymatic nucleic acid molecules. Arrow indicates the site ofcleavage. --------- indicate the target sequence. Lines interspersedwith dots are meant to indicate tertiary interactions.—is meant toindicate base-paired interaction. Group I Intron: P1-P9.0 representvarious stem-loop structures (Cech et al., 1994, Nature Struc. Bio., 1,273). RNase P (M1RNA): EGS represents external guide sequence (Forsteret al., 1990, Science, 249, 783; Pace et al., 1990, J. Biol. Chem., 265,3587). Group II Intron: 5′SS means 5′ splice site; 3′SS means 3′-splicesite; IBS means intron binding site; EBS means exon binding site (Pyleet al., 1994, Biochemistry, 33, 2716). VS RNA: I-VI are meant toindicate six stem-loop structures; shaded regions are meant to indicatetertiary interaction (Collins, International PCT Publication No. WO96/19577). HDV Ribozyme: : I-IV are meant to indicate four stem-loopstructures (Been et al., U.S. Pat. No. 5,625,047). Hammerhead Ribozyme::I-III are meant to indicate three stem-loop structures; stems I-III canbe of any length and may be symmetrical or asymmetrical (Usman et al.,1996, Curr. Op. Struct. Bio., 1, 527). Hairpin Ribozyme: Helix 1, 4 and5 can be of any length; Helix 2 is between 3 and 8 base-pairs long; Y isa pyrimidine; Helix 2 (H2) is provided with a least 4 base pairs (i.e.,n is 1, 2, 3 or 4) and helix 5 can be optionally provided of length 2 ormore bases (preferably 3-20 bases, i.e., m is from 1-20 or more). Helix2 and helix 5 may be covalently linked by one or more bases (i.e., r is≧1 base). Helix 1, 4 or 5 may also be extended by 2 or more base pairs(e.g., 4-20 base pairs) to stabilize the ribozyme structure, andpreferably is a protein binding site. In each instance, each N and N′independently is any normal or modified base and each dash represents apotential base-pairing interaction. These nucleotides may be modified atthe sugar, base or phosphate. Complete base-pairing is not required inthe helices, but is preferred. Helix 1 and 4 can be of any size (i.e., oand p is each independently from 0 to any number, e.g., 20) as long assome base-pairing is maintained. Essential bases are shown as specificbases in the structure, but those in the art will recognize that one ormore may be modified chemically (abasic, base, sugar and/or phosphatemodifications) or replaced with another base without significant effect.Helix 4 can be formed from two separate molecules, i.e., without aconnecting loop. The connecting loop when present may be aribonucleotide with or without modifications to its base, sugar orphosphate. “q” is ≧2 bases. The connecting loop can also be replacedwith a non-nucleotide linker molecule. H refers to bases A, U, or C. Yrefers to pyrimidine bases. “_” refers to a covalent bond. (Burke etal., 1996, Nucleic Acids & Mol. Biol., 10, 129; Chowrira et al., U.S.Pat. No. 5,631,359).

[0051]FIG. 2 depicts a scheme for the synthesis of a xylo ribonucleosidephosphoramidite.

[0052]FIG. 3 is a diagrammatic representation of hammerhead (HH)ribozyme targeted against stromelysin RNA (site 617) with variousmodifications.

[0053] Synthesis of Polynucleotides

[0054] Synthesis of polynucleotides greater than 100 nucleotides inlength is difficult using automated methods, and the therapeutic cost ofsuch molecules is prohibitive. In this invention, small enzymaticnucleic acid motifs (e.g., of the hammerhead or the hairpin structure)are used for exogenous delivery. The simple structure of these moleculesincreases the ability of the enzymatic nucleic acid to invade targetedregions of the mRNA structure.

[0055] By “polynucleotide” as used herein is meant a molecule having twoor more nucleotides. The polynucleotide can be single, double ormultiple stranded and may comprise modified or unmodified nucleotides ornon-nucleotides or various mixtures and combinations thereof.

[0056] RNA molecules, such as the ribozymes are chemically synthesized.The method of synthesis used follows the procedure for normal RNAsynthesis as described in Usman et al., 1987 J. Am. Chem. Soc., 109,7845; Scaringe et al., 1990 Nucleic Acids Res., 18, 5433; and Wincott eta., 1995 Nucleic Acids Res. 23, 2677-2684 and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. Small scale synthesiswere conducted on a 394 Applied Biosystems, Inc. synthesizer using amodified 2.5 μmol scale protocol with a 5 min coupling step foralkylsilyl protected nucleotides and 2.5 min coupling step for2′-O-methylated nucleotides. Table II outlines the amounts, and thecontact times, of the reagents used in the synthesis cycle. A 6.5-foldexcess (163 μL of 0.1 M=16.3 μmol) of phosphoramidite and a 24-foldexcess of S-ethyl tetrazole (238 μL of 0.25 M=59.5 μmol) relative topolymer-bound 5′-hydroxyl was used in each coupling cycle. Averagecoupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by calorimetric quantitation of the trityl fractions, were97.5-99%. Other oligonucleotide synthesis reagents for the 394 AppliedBiosystems, Inc. synthesizer: detritylation solution was 2% TCA inmethylene chloride (ABI); capping was performed with 16% N-methylimidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF(ABI); oxidation solution was 16.9 mM I₂, 49 mM pyridine, 9% water inTHF (Millipore). B & J Synthesis Grade acetonitrile was used directlyfrom the reagent bottle. S-Ethyl tetrazole solution (0.25 M inacetonitrile) was made up from the solid obtained from AmericanInternational Chemical, Inc.

[0057] Deprotection of the RNA was performed as follows. Thepolymer-bound oligoribonucleotide, trityl-off, was transferred from thesynthesis column to a 4 mL glass screw top vial and suspended in asolution of methylamine (MA) at 65° C. for 10 min. After cooling to −20°C., the supernatant was removed from the polymer support. The supportwas washed three times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed andthe supernatant was then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, were dried to a whitepowder.

[0058] The base-deprotected oligoribonucleotide was resuspended inanhydrous TEA.HF/NMP solution (250 μL of a solution of 1.5 mLN-methylpyrrolidinone, 750 μL TEA and 1.0 mL TEA.3HF to provide a 1.4MHF concentration) and heated to 65° C. for 1.5 h. The resulting, fullydeprotected, oligomer was quenched with 50 mM TEAB (9 mL) prior to anionexchange desalting.

[0059] For anion exchange desalting of the deprotected oligomer, theTEAB solution was loaded onto a Qiagen 500® anion exchange cartridge(Qiagen Inc.) that was prewashed with 50 mM TEAB (10 mL). After washingthe loaded cartridge with 50 mM TEAB (10 mL), the RNA was eluted with 2M TEAB (10 mL) and dried down to a white powder.

[0060] RNAs are purified by gel electrophoresis using general methods orare purified by high pressure liquid chromatography (HPLC; SeeStinchcomb et al., International PCT Publication No. WO 95/23225, thetotality of which is hereby incorporated herein by reference) and areresuspended in water.

[0061] Enzymatic Nucleic Acid Molecules

[0062] The enzymatic nucleic acid is able to intramolecularly orintermolecularly cleave RNA or DNA and thereby inactivate a target RNAor DNA molecule. The enzymatic nucleic acid molecule that hascomplementarity in a substrate binding region to a specified genetarget, also has an enzymatic activity that specifically cleaves RNA orDNA in that target. This complementarity functions to allow sufficienthybridization of the enzymatic RNA molecule to the target RNA or DNA toallow the cleavage to occur. 100% Complementarity is preferred, butcomplementarity as low as 50-75% may also be useful in this invention.The nucleic acids may be modified at the base, sugar, and/or phosphategroups.

[0063] The term enzymatic nucleic acid is used interchangeably withphrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA,nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, minizyme, leadzyme,oligozyme, or DNA enzyme.

[0064] By “complementarity” is meant a nucleic acid that can formhydrogen bond(s) with other RNA sequence by either traditionalWatson-Crick or other non-traditional types (for example, Hoogsteentype) of base-paired interactions.

[0065] Nucleic acid molecules having an endonuclease enzymatic activityare able to repeatedly cleave other separate RNA molecules in anucleotide base sequence-specific manner. Such enzymatic RNA moleculescan be targeted to virtually any RNA transcript, and efficient cleavageachieved in vitro (Zaug et al., 324, Nature 429 1986; Kim et al., 84Proc. Natl. Acad. Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 NucleicAcids Research 1371, 1989).

[0066] Because of their sequence-specificity, trans-cleaving ribozymesshow promise as therapeutic agents for human disease (Usman & McSwiggen,1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J.Med. Chem. 38, 2023-2037). Ribozymes can be designed to cleave specificRNA targets within the background of cellular RNA. Such a cleavage eventrenders the mRNA non-functional and abrogates protein expression fromthat RNA. In this manner, synthesis of a protein associated with adisease state can be selectively inhibited.

[0067] Seven basic varieties of naturally-occurring enzymatic RNAs areknown presently. Each can catalyze the hydrolysis of RNA phosphodiesterbonds in trans (and thus can cleave other RNA molecules) underphysiological conditions. Table I summarizes some of the characteristicsof these ribozymes. In general, enzymatic nucleic acids act by firstbinding to a target RNA. Such binding occurs through the target bindingportion of a enzymatic nucleic acid which is held in close proximity toan enzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

[0068] The enzymatic nature of a ribozyme is advantageous over othertechnologies, since the effective concentration of ribozyme necessary toeffect a therapeutic treatment is lower than that of an antisenseoligonucleotide. This advantage reflects the ability of the ribozyme toact enzymatically. Thus, a single ribozyme molecule is able to cleavemany molecules of target RNA. In addition, the ribozyme is a highlyspecific inhibitor, with the specificity of inhibition depending notonly on the base-pairing mechanism of binding, but also on the mechanismby which the molecule inhibits the expression of the RNA to which itbinds. That is, the inhibition is caused by cleavage of the RNA targetand so specificity is defined as the ratio of the rate of cleavage ofthe targeted RNA over the rate of cleavage of non-targeted RNA. Thiscleavage mechanism is dependent upon factors additional to thoseinvolved in base-pairing. Thus, it is thought that the specificity ofaction of a ribozyme is greater than that of antisense oligonucleotidebinding the same RNA site.

[0069] In one aspect, an enzymatic nucleic acid molecule is formed in ahammerhead (see for example FIG. 1 and 2) or hairpin motif (FIG. 1), butmay also be formed in the motif of a hepatitis delta virus (HDV), groupI intron, RNaseP RNA (in association with an external guide sequence) orNeurospora VS RNA (FIG. 1). Examples of such hammerhead motifs aredescribed by Rossi et al., 1992, Aids Research and Human Retroviruses 8,183; Usman et al., 1996, Curr. Op. Struct. Biol., 1, 527; of hairpinmotifs by Hampel et al., EP 0360257; Hampel and Tritz, 1989 Biochemistry28, 4929; and Hampel et al., 1990 Nucleic Acids Res. 18, 299; Chowriraet al., U.S. Pat. No. 5,631,359; an example of the hepatitis delta virusmotif is described by Perrotta and Been, 1992 Biochemistry 31, 16; Beenet al., U.S. Pat. No. 5,625,047; of the RNaseP motif by Guerrier-Takadaet al., 1983 Cell 35, 849; Forster and Altman, 1990 Science 249, 783;Neurospora VS RNA ribozyme motif is described by Collins (Saville andCollins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl.Acad. Sci. USA 88, 8826-8830; Guo and Collins, 1995 EMBO J. 14, 368) andof the Group I intron by Zaug et al., 1986, Nature, 324, 429; Cech etal., U.S. Pat. No. 4,987,071. (All these publications are herebyincorporated by references herein.) These specific motifs are notlimiting in the invention and those skilled in the art will recognizethat all that is important in an enzymatic nucleic acid molecule withendonuclease activity of this invention is that it has a specificsubstrate binding site which is complementary to one or more of thetarget gene RNA and that it have nucleotide sequences within orsurrounding that substrate binding site which impart an RNA cleavingactivity to the molecule. The length of the binding site varies fordifferent ribozyme motifs, and a person skilled in the art willrecognize that to achieve an optimal ribozyme activity the length of thebinding arm should be of sufficient length to form a stable interactionwith the target nucleic acid sequence.

[0070] Catalytic activity of the ribozymes described in the instantinvention can be optimized as described by Draper et al., supra. Thedetails will not be repeated here, but include altering the length ofthe ribozyme binding arms, or chemically synthesizing ribozymes withmodifications (base, sugar and/or phosphate) that prevent theirdegradation by serum ribonucleases and/or enhance their enzymaticactivity (see e.g., Eckstein et al., International Publication No. WO92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991Science 253, 314; Usman and Cedergren, 1992 Trends in Biochem. Sci. 17,334; Usman et al., International Publication No. WO 93/15187; and Rossiet al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; and Burgin et al., supra; all of these describe variouschemical modifications that can be made to the base, phosphate and/orsugar moieties of enzymatic RNA molecules). Modifications which enhancetheir efficacy in cells, and removal of bases from stem loop structuresto shorten RNA synthesis times and reduce chemical requirements aredesired. (All these publications are hereby incorporated by referenceherein).

[0071] There are several examples in the art describing sugar andphosphate modifications that can be introduced into enzymatic nucleicacid molecules without significantly effecting catalysis and withsignificant enhancement in their nuclease stability and efficacy.Ribozymes are modified to enhance stability and/or enhance catalyticactivity by modification with nuclease resistant groups, for example,2′-amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H, nucleotide basemodifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34;Usman et al., 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996Biochemistry 35, 14090). Sugar modification of enzymatic nucleic acidmolecules have been extensively described in the art (see Eckstein etal., International Publication PCT No. WO 92/07065; Perrault et al.Nature 1990, 344, 565-568; Pieken et al. Science 1991, 253, 314-317;Usman and Cedergren, Trends in Biochem. Sci. 1992, 17, 334-339; Usman etal. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No.5,334,711 and Beigelman et al., 1995 J. Biol. Chem. 270, 25702; all ofthe references are hereby incorporated in their totality by referenceherein).

[0072] Such publications describe general methods and strategies todetermine the location of incorporation of sugar, base and/or phosphatemodifications and the like into ribozymes without inhibiting catalysis,and are incorporated by reference herein. In view of such teachings,similar modifications can be used as described herein to modify thenucleic acid catalysts of the instant invention.

[0073] Nucleic acid catalysts having chemical modifications whichmaintain or enhance enzymatic activity are provided. Such nucleic acidis also generally more resistant to nucleases than unmodified nucleicacid. Thus, in a cell and/or in vivo the activity may not besignificantly lowered. As exemplified herein such ribozymes are usefulin a cell and/or in vivo even if activity over all is reduced 10 fold(Burgin et al., 1996, Biochemistry, 35, 14090). Such ribozymes hereinare said to “maintain” the enzymatic activity on all RNA ribozyme.

[0074] Therapeutic ribozymes delivered exogenously must optimally bestable within cells until translation of the target RNA has beeninhibited long enough to reduce the levels of the undesirable protein.This period of time varies between hours to days depending upon thedisease state. Clearly, ribozymes must be resistant to nucleases inorder to function as effective intracellular therapeutic agents.Improvements in the chemical synthesis of RNA (Wincott et al., 1995Nucleic Acids Res. 23, 2677; incorporated by reference herein) haveexpanded the ability to modify ribozymes by introducing nucleotidemodifications to enhance their nuclease stability as described above.

[0075] By “nucleotide” as used herein is as recognized in the art toinclude natural bases (standard), and modified bases well known in theart. Such bases are generally located at the 1′ position of a sugarmoiety. Nucleotide generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;all hereby incorporated by reference herein). There are several examplesof modified nucleic acid bases known in the art and has recently beensummarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some ofthe non-limiting examples of base modifications that can be introducedinto enzymatic nucleic acids without significantly effecting theircatalytic activity include, inosine, purine, pyridin-4-one,pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g.,5-methylcytidine), 5-alkyluridines (e.g. ribothymidine), 5-halouridine(e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine) and others (Burgin et al., 1996, Biochemistry, 35,14090). By “modified bases” in this aspect is meant nucleotide basesother than adenine, guanine, cytosine and uracil at 1′ position or theirequivalents; such bases may be used within the catalytic core of theenzyme and/or in the substrate-binding regions.

[0076] By “unmodified nucleoside” is meant one of the bases adenine,cytosine, guanine, uracil joined to the 1′ carbon of b-D-ribo-furanose.

[0077] By “modified nucleoside” is meant any nucleotide base whichcontains a modification in the chemical structure of an unmodifiednucleotide base, sugar and/or phosphate.

[0078] Various modifications to ribozyme structure can be made toenhance the utility of ribozymes. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch ribozymes to the target site, eg., to enhance penetration ofcellular membranes, and confer the ability to recognize and bind totargeted cells.

[0079] Administration of Polynucleotides

[0080] Sullivan et al., PCT WO 94/02595, describes the general methodsfor delivery of enzymatic RNA molecules. Ribozymes may be administeredto cells by a variety of methods known to those familiar to the art,including, but not restricted to, encapsulation in liposomes, byiontophoresis, or by incorporation into other vehicles, such ashydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesivemicrospheres. For some indications, ribozymes may be directly deliveredex vivo to cells or tissues with or without the aforementioned vehicles.Alternatively, the RNA/vehicle combination is locally delivered bydirect injection or by use of a catheter, infusion pump or stent. Otherroutes of delivery include, but are not limited to, intravascular,intramuscular, subcutaneous or joint injection, aerosol inhalation, oral(tablet or pill form), topical, systemic, ocular, intraperitoneal and/orintrathecal delivery. More detailed descriptions of ribozyme deliveryand administration are provided in Sullivan et al., supra and Draper etal., PCT W093/23569 which have been incorporated by reference herein.

[0081] The molecules of the instant invention can be used aspharmaceutical agents. Pharmaceutical agents prevent, inhibit theoccurrence, or treat (alleviate a symptom to some extent, preferably allof the symptoms) of a disease state in a patient.

[0082] By “patient” is meant an organism which is a donor or recipientof explanted cells or the cells themselves. “Patient” also refers to anorganism to which the compounds of the invention can be administered.Preferably, a patient is a mammal, e.g., a human, primate or a rodent.

[0083] The negatively charged polynucleotides of the invention can beadministered (e.g., RNA, DNA or protein) and introduced into a patientby any standard means, with or without stabilizers, buffers, and thelike, to form a pharmaceutical composition. When it is desired to use aliposome delivery mechanism, standard protocols for formation ofliposomes can be followed. The compositions of the present invention mayalso be formulated and used as tablets, capsules or elixirs for oraladministration; suppositories for rectal administration; sterilesolutions; suspensions for injectable administration; and the like.

[0084] The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

[0085] A pharmacological composition or formulation refers to acomposition or formulation in a form suitable for administration, e.g.,systemic administration, into a cell or patient, preferably a human.Suitable forms, in part, depend upon the use or the route of entry, forexample oral, transdermal, or by injection. Such forms should notprevent the composition or formulation to reach a target cell (i.e., acell to which the negatively charged polymer is desired to be deliveredto). For example, pharmacological compositions injected into the bloodstream should be soluble. Other factors are known in the art, andinclude considerations such as toxicity and forms which prevent thecomposition or formulation from exerting its effect.

[0086] By “systemic administration” is meant in vivo systemic absorptionor accumulation of drugs in the blood stream followed by distributionthroughout the entire body. Administration routes which lead to systemicabsorption include, without limitations: intravenous, subcutaneous,intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.Each of these administration routes expose the desired negativelycharged polymers, e.g., nucleic acids, to an accessible diseased tissue.The rate of entry of a drug into the circulation has been shown to be afunction of molecular weight or size. The use of a liposome or otherdrug carrier comprising the compounds of the instant invention canpotentially localize the drug, for example, in certain tissue types,such as the tissues of the reticular endothelial system (RES). Aliposome formulation which can facilitate the association of drug withthe surface of cells, such as, lymphocytes and macrophages is alsouseful. This approach may provide enhanced delivery of the drug totarget cells by taking advantage of the specificity of macrophage andlymphocyte immune recognition of abnormal cells, such as the cancercells.

[0087] The invention also features the use of the a compositioncomprising surface-modified liposomes containing poly (ethylene glycol)lipids (PEG-modified, or long-circulating liposomes or stealthliposomes). These formulations offer an method for increasing theaccumulation of drugs in target tissues. This class of drug carriersresists opsonization and elimination by the mononuclear phagocyticsystem (MPS or RES), thereby enabling longer blood circulation times andenhanced tissue exposure for the encapsulated drug (Lasic et al. Chem.Rev. 1995, 95, 2601-2627; Ishiwataet al., Chem. Pharm. Bull. 1995, 43,1005-1011). Such liposomes have been shown to accumulate selectively intumors, presumably by extravasation and capture in the neovascularizedtarget tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al.,1995, Biochim. Biophys. Acta, 1238, 86-90). The long-circulatingliposomes enhance the pharmacokinetics and pharmacodynamics of DNA andRNA, particularly compared to conventional cationic liposomes which areknown to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem.1995, 42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392; all ofthese are incorporated by reference herein). Long-circulating liposomesare also likely to protect drugs from nuclease degradation to a greaterextent compared to cationic liposomes, based on their ability to avoidaccumulation in metabolically aggressive MPS tissues such as the liverand spleen. All of these references are incorporated by referenceherein.

[0088] The present invention also includes compositions prepared forstorage or administration which include a pharmaceutically effectiveamount of the desired compounds in a pharmaceutically acceptable carrieror diluent. Acceptable carriers or diluents for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985) hereby incorporated by reference herein. For example,preservatives, stabilizers, dyes and flavoring agents may be provided.Id. at 1449. These include sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. In addition, antioxidants and suspending agentsmay be used. Id.

[0089] A pharmaceutically effective dose is that dose required toprevent, inhibit the occurrence, or treat (alleviate a symptom to someextent, preferably all of the symptoms) of a disease state. Thepharmaceutically effective dose depends on the type of disease, thecomposition used, the route of administration, the type of mammal beingtreated, the physical characteristics of the specific mammal underconsideration, concurrent medication, and other factors which thoseskilled in the medical arts will recognize. Generally, an amount between0.1 mg/kg and 100 mg/kg body weight/day of active ingredients isadministered dependent upon potency of the negatively charged polymer.

EXAMPLES

[0090] The following are non-limiting examples showing the synthesis andactivity of the certain compounds of Formula I of the instant inventionand polynucleotides comprising one or more of these compounds. Those inthe art will recognize that certain reaction conditions such astemperatures, pH, ionic conditions, reaction times and solventconditions described in the following examples are not meant to belimiting and can be readily modified without significantly effecting thesynthesis.

Example 1 Synthesis of Xylo Nucleoside Phosphoramidite

[0091] Preparation of Protected 1-(β-D-xylopentofuranosyl)nucleoside

[0092] Referring to FIG. 2, 2′-O-TBDMS-5′-O-DMT ribonucleoside 1 (2mmol) was added to a solution of CrO₃ (600 mg), pyridine (1 ml) andacetic anhydride (0.6 ml) in CH₂Cl₂ (15 ml) and the reaction mixturestirred at room temperature for 1 hour. Ethyl acetate (100 ml) was thenadded and the mixture filtered through a Celite pad. The filtrate wasconcentrated in vacuo (40° C.), ethyl acetate (100 ml) was added and themixture filtered slowly through the mixture of silica gel and Florisil(1:1, 40 g). The filtrate was concentrated in vacuo (40° C.) and useddirectly in the next step.

[0093] The above material was dissolved in ethanol (30 ml) andNaB(OAc)₃H (848 mg, 2 eq) was added. The reaction mixture was stirred atroom temperature overnight and the solvent removed in vacuo. The residuewas partitioned between ethyl acetate and brine, organic layer waswashed with aqueous 5% NaHCO₃ solution, dried (Na₂SO₄) and evaporated toa colorless foam. Purification by flash silica gel column chromatographyusing CH₂Cl₂/MeOH or CH₂CH₂Cl₂/THF mixtures yielded pure products(scheme 1, 2) in 60-75% yield (based on the starting ribo nucleosides).

[0094] Preparation of 3′-O-phosphoramidites

[0095] 3′-O-Phosphoramidites 3 were prepared in 65-70(G) or-75-85(A) %yield using the standard phosphitylation procedure (Tuschl, T., et al.Biochemistry 1993, 32, 11658-11668).

[0096] This scheme can be used to synthesize xylo nucleosidephosphoramidites such as xyloadenosine, xyloguanosine, xylouridine,xylocytidine and others.

Example 2 Incorporation of Phosphoramidites into Ribozymes

[0097] The above monomers 3 were incorporated into ribozymes usingstandard procedures (Wincott, et al. Nucleic Acids Res 1995, 23,2677-2684; Usman et al., J. Am. Chem. Soc. 1987, 109, 7845-7854;Scaringe et al., Nucleic Acids Res. 1990, 18, 5433-5441) and makes useof common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. Theaverage stepwise coupling yields were >98%. These nucleotides may beincorporated not only into hammerhead ribozymes, but also into hairpin,VS ribozymes, hepatitis delta virus, or Group I or Group II introns.They are, therefore, of general use as replacement motifs in any nucleicacid structure. The coupling time for the incorporation of modifiedphosphoramidites was extended to 20 minutes (Seela, F., et al. 1 Helv.Chim. Acta 1994, 77, 883-895). Examples of ribozyme synthesizedaccording to this invention are shown in FIG. 3.

Example 3 Cleavage of Short Substrate Using Xylo Modified Ribozymes

[0098] Ribozyme Reactions

[0099] Ribozyme (1 μM) was incubated in 50 mM Tris (pH 8.0) and 10 mMMgCl₂ at 37° C. with trace amounts of short substrate (>1 nMol of RNA).Reaction times were modulated to give accurate kinetics of cleavagevalues. Ribozyme with xylo A residue at A15.1 and/or A6 demonstrated thesame cleavage activity as parent “5-Ribo” motif (Table IIIA).Incorporation of xylo G at G12 is also tolerated though cleavageactivity is reduced by 5 fold (Table IIIB).

[0100] Diagnostic Uses

[0101] Ribozymes of this invention may be used as diagnostic tools toexamine genetic drift and mutations within diseased cells or to detectthe presence of a specific RNA in a cell. The close relationship betweenribozyme activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple ribozymes described in this invention, one may map nucleotidechanges which are important to RNA structure and function in vitro, aswell as in cells and tissues. Cleavage of target RNAs with ribozymes maybe used to inhibit gene expression and define the role (essentially) ofspecified gene products in the progression of disease. In this manner,other genetic targets may be defined as important mediators of thedisease. These experiments will lead to better treatment of the diseaseprogression by affording the possibility of combinational therapies(e.g., multiple ribozymes targeted to different genes, ribozymes coupledwith known small molecule inhibitors, or intermittent treatment withcombinations of ribozymes and/or other chemical or biologicalmolecules). Other in vitro uses of ribozymes of this invention are wellknown in the art, and include detection of the presence of mRNAsassociated with related condition. Such RNA is detected by determiningthe presence of a cleavage product after treatment with a ribozyme usingstandard methodology.

[0102] In a specific example, ribozymes which can cleave only wild-typeor mutant forms of the target RNA are used for the assay. The firstribozyme is used to identify wild-type RNA present in the sample and thesecond ribozyme will be used to identify mutant RNA in the sample. Asreaction controls, synthetic substrates of both wild-type and mutant RNAwill be cleaved by both ribozymes to demonstrate the relative ribozymeefficiencies in the reactions and the absence of cleavage of the“non-targeted” RNA species. The cleavage products from the syntheticsubstrates will also serve to generate size markers for the analysis ofwild-type and mutant RNAs in the sample population. Thus each analysiswill require two ribozymes, two substrates and one unknown sample whichwill be combined into six reactions. The presence of cleavage productswill be determined using an RNAse protection assay so that full-lengthand cleavage fragments of each RNA can be analyzed in one lane of apolyacrylamide gel. It is not absolutely required to quantify theresults to gain insight into the expression of mutant RNAs and putativerisk of the desired phenotypic changes in target cells. The expressionof mRNA whose protein product is implicated in the development of thephenotype is adequate to establish risk. If probes of comparablespecific activity are used for both transcripts, then a qualitativecomparison of RNA levels will be adequate and will decrease the cost ofthe initial diagnosis. Higher mutant form to wild-type ratios will becorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

[0103] Additional Uses

[0104] Nucleic acid molecules of the instant invention might have manyof the same applications for the study of RNA that DNA restrictionendonucleases have for the study of DNA (Nathans et al., 1975 Ann. Rev.Biochem. 44:273). For example, the pattern of restriction fragmentscould be used to establish sequence relationships between two relatedRNAs, and large RNAs could be specifically cleaved to fragments of asize more useful for study. The ability to engineer sequence specificityof the ribozyme is ideal for cleavage of RNAs of unknown sequence.Nucleic acid molecules (e.g., ribozymes) of the invention can be used,for example, to target cleavage of virtually any RNA transcript (Zaug etal., 324, Nature 429 1986; Cech, 260 JAMA 3030, 1988; and Jefferies etal., 17 Nucleic Acids Research 1371, 1989). Such nucleic acids can beused as a therapeutic or to validate a therapeutic gene target and/or todetermine the function of a gene in a biological system (Christoffersen,1997, Nature Biotech. 15, 483).

[0105] Various ligands can be attached to oligonucleotides using thecomponds of Formula I for the purposes of cellular delivery, nucleaseresistance, cellular trafficking and localization, chemical ligation ofoligonucleotide fragments. Incorporation of one or more compounds ofFormula I into a ribozyme may increase its effectiveness. Compounds ofFormula I can be used as potential antiviral agents.

[0106] Other embodiments are within the following claims. TABLE ICharacteristics of naturally occurring ribozymes Group I Introns Size:˜150 to >1000 nucleotides. Requires a U in the target sequenceimmediately 5′ of the cleavage site. Binds 4-6 nucleotides at the5′-side of the cleavage site. Reaction mechanism: attack by the 3′-OH ofguanosine to generate cleavage products with 3′-OH and 5′-guanosine.Additional protein cofactors required in some cases to help folding andmaintainance of the active structure. Over 300 known members of thisclass. Found as an intervening sequence in Tetrahymena thermophila rRNA,fungal mitochondria, chloroplasts, phage T4, blue-green algae, andothers. Major structural features largely established throughphylogenetic comparisons, mutagenesis, and biochemical studies[^(i),^(ii)] Complete kinetic framework established for one ribozyme[^(iii),^(iv),^(v),^(vi)]. Studies of ribozyme folding and substratedocking underway [^(vii),^(viii),^(ix)]. Chemical modificationinvestigation of important residues well established [^(x),^(xi)]. Thesmall (4-6 nt) binding site may make this ribozyme too non-specific fortargeted RNA cleavage, however, the Tetrahymena group I intron has beenused to repair a “defective” β-galactosidase message by the ligation ofnew β-galactosidase sequences onto the defective message [^(xii)]. RNAseP RNA (M1 RNA) Size: ˜290 to 400 nucleotides. RNA portion of aubiquitous ribonucleoprotein enzyme. Cleaves tRNA precursors to formmature tRNA [^(xiii)]. Reaction mechanism: possible attack by M²⁺-OH togenerate cleavage products with 3′-OH and 5′-phosphate. RNAse P is foundthroughout the prokaryotes and eukaryotes. The RNA subunit has beensequenced from bacteria, yeast, rodents, and primates. Recruitment ofendogenous RNAse P for therapeutic applications is possible throughhybridization of an External Guide Sequence (EGS) to the target RNA[^(xiv),^(xv)] Important phosphate and 2′ OH contacts recentlyidentified [^(xvi),^(xvii)] Group II Introns Size: >1000 nucleotides.Trans cleavage of target RNAs recently demonstrated [^(xviii),^(xix)].Sequence requirements not fully determined. Reaction mechanism: 2′-OH ofan internal adenosine generates cleavage products with 3′-OH and a“lariat” RNA containing a 3′-5′ and a 2′-5′ branch point. Only naturalribozyme with demonstrated participation in DNA cleavage [^(xx),^(xxi)]in addition to RNA cleavage and ligation. Major structural featureslargely established through phylogenetic comparisons [^(xxii)].Important 2′ OH contacts beginning to be identified [^(xxiii)] Kineticframework under development [^(xxiv)] Neurospora VS RNA Size: ˜144nucleotides. Trans cleavage of hairpin target RNAs recently demonstrated[^(xxv)]. Sequence requirements not fully determined. Reactionmechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavageproducts with 2′,3′-cyclic phosphate and 5′-OH ends. Binding sites andstructural requirements not fully determined. Only 1 known member ofthis class. Found in Neurospora VS RNA. Hammerhead Ribozyme (see textfor references) Size: ˜13 to 40 nucleotides. Requires the targetsequence UH immediately 5′ of the cleavage site. Binds a variable numbernucleotides on both sides of the cleavage site. Reaction mechanism:attack by 2′-OH 5′ to the scissile bond to generate cleavage productswith 2′,3′-cyclic phosphate and 5′-OH ends. 14 known members of thisclass. Found in a number of plant pathogens (virusoids) that use RNA asthe infectious agent. Essential structural features largely defined,including 2 crystal structures [^(xxvi),^(xxvii)] Minimal ligationactivity demonstrated (for engineering through in vitro selection)[^(xxviii)] Complete kinetic framework established for two or moreribozymes [^(xxix)]. Chemical modification investigation of importantresidues well established [^(xxx)]. Hairpin Ribozyme Size: ˜50nucleotides. Requires the target sequence GUC immediately 3′ of thecleavage site. Binds 4-6 nucleotides at the 5′-side of the cleavage siteand a variable number to the 3′-side of the cleavage site. Reactionmechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavageproducts with 2′,3′-cyclic phosphate and 5′-OH ends. 3 known members ofthis class. Found in three plant pathogen (satellite RNAs of the tobaccoringspot virus, arabis mosaic virus and chicory yellow mottle virus)which uses RNA as the infectious agent. Essential structural featureslargely defined [^(xxxi),^(xxxii),^(xxxiii),^(xxxiv)] Ligation activity(in addition to cleavage activity) makes ribozyme amenable toengineering through in vitro selection [^(xxxv)] Complete kineticframework established for one ribozyme [^(xxxvi)]. Chemical modificationinvestigation of important residues begun [^(xxxvii),^(xxxviii)].Hepatitis Delta Virus (HDV) Ribozyme Size: ˜60 nucleotides. Transcleavage of target RNAs demonstrated [^(xxxix)]. Binding sites andstructural requirements not fully determined, although no sequences 5′of cleavage site are required. Folded ribozyme contains a pseudoknotstructure [^(xl)]. Reaction mechanism: attack by 2′-OH 5′ to thescissile bond to generate cleavage products with 2′,3′-cyclic phosphateand 5′-OH ends. Only 2 known members of this class. Found in human HDV.Circular form of HDV is active and shows increased nuclease stability[^(xli)]

[0107] TABLE II 2.5 μmol RNA Synthesis Cycle Wait Reagent EquivalentsAmount Time* Phosphoramidites 6.5 163 μL 2.5 S-Ethyl Tetrazole 23.8 238μL 2.5 Acetic Anhydride 100 233 μL  5 sec N-Methyl Imidazole 186 233 μL 5 sec TCA 83.2 1.73 mL 21 sec Iodine 8.0 1.18 mL 45 sec Acetonitrile NA6.67 mL NA

[0108] TABLE IIIA Ribozyme K_(obs) (min⁻¹) K_(rel) A 4 1 B 0.7 0.2 C0.002 0.0005 D 0.004 0.001

[0109] TABLE IIIB Ribozyme K_(obs) (min⁻¹) K_(rel) A 0.32938 1.00 E0.29395 0.89 F 0.22249 0.68 G 0.28201 0.86

1. A compound having the formula I:

wherein, R₁ is OH, O—R₃, wherein R₃ is independently a moiety selected from a group consisting of alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester, C—R₃, wherein R₃ is independently a moiety selected from a group consisting of alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester, halo, NHR₄ wherein R₄ is independently a moiety selected from a group consisting of alkyl (C1-22), acyl (C1-22), substituted or unsubstituted aryl), or OCH₂SCH₃ (methylthiomethyl), ONHR₅ where R₅ is independently a moiety selected from a group consisting of H, aminoacyl group, peptidyl group, biotinyl group, cholesteryl group, lipoic acid residue, retinoic acid residue, folic acid residue, ascorbic acid residue, nicotinic acid residue, 6-aminopenicillanic acid residue, 7-aminocephalosporanic acid residue, alkyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide or ester, ON═R₆, where R₆ is independently pyridoxal residue, pyridoxal-5-phosphate residue, 13-cis-retinal residue, 9-cis-retinal residue, alkyl, alkenyl, alkynyl, alkylaryl, carbocyclic alkylaryl, or heterocyclic alkylaryl; B is independently a nucleotide base or its analog or hydrogen; X is independently a phosphorus-containing group; and R₂ is independently blocking group or a phosphorus-containing group.
 2. The compound of claim 1, wherein said compound is a nucleotide.
 3. The compound of claim 1, wherein said compound is a nucleotide-tri-phosphate.
 4. A polynucleotide comprising the compound of claim 1 at one or more positions.
 5. The polynucleotide of claim 4, wherein said polynucleotide is an enzymatic nucleic acid.
 6. The enzymatic nucleic acid of claim 5, wherein said nucleic acid is in a hammerhead configuration.
 7. The enzymatic nucleic acid of claim 6, wherein said nucleic acid is in a hairpin configuration.
 8. The enzymatic nucleic acid of claim 6, wherein said nucleic acid is in a hepatitis delta virus, group I intron, VS RNA, group II intron or RNase P RNA configuration.
 9. The compound of claim 1, wherein said compound is xylo riboadenosine.
 10. The compound of claim 1, wherein said compound is xylo riboguanosine.
 11. The compound of claim 1, wherein said compound is xylo ribonucleoside phosphoramidite.
 12. The compound of claim 1, wherein said compound is xylo riboguanosine phosphoramidite.
 13. The compound of claim 11, wherein said compound is xylo riboadenosine phosphoramidite.
 14. A mammalian cell comprising the compound of claim
 1. 15. The mammalian cell of claim 14, wherein said cell is a human cell.
 16. A mammalian cell comprising the compound of claim
 5. 17. The mammalian cell of claim 16, wherein said cell is a human cell.
 18. A method of making a polynucleotide of claim
 4. 19. A method of modulating gene expression using a polynucleotide of claim
 4. 20. A pharmaceutical composition comprising a compound of claim
 1. 21. A pharmaceutical composition comprising a polynucleotide of claim
 5. 22. The compound of claim 1, wherein said compound is used as an antiviral agent.
 23. A process for the synthesis of a xylo ribonucleoside phosphoramidite comprising the steps of: a) oxidation of a 2′ and 5′-protected ribonucleoside using an oxidant followed by reduction using a reducing agent under conditions suitable for the formation of 2′ and 5′-protected xylofuranosyl nucleoside; and b) phosphitylation under conditions suitable for the formation of xylofuranosyl nucleoside phosphoramidite.;
 24. The process of claim 23, wherein said oxidation is carried out in the presence of chromium oxide, pyridine, and aceticanhydride.
 25. The process of claim 23, wherein said oxidation is carried out in the presence of dimethylsulfoxide and aceticanhydride.
 26. The process of claim 23, wherein said oxidation is carried out in the presence of Dess-Martin reagent (periodinane).
 27. The process of claim 23, wherein said reduction is carried out in the presence of triacetoxy sodium borohydride.
 28. The process of claim 23, wherein said reduction is carried out in the presence of sodium borohydride
 29. The process of claim 23, wherein said reduction is carried out in the presence of lithium borohydride, 